Engineered anomaterials including carbon nanotubes (CNTs) have emerged as one of the most important classes of new materials having enormous potential to create new and better products. Accumulating evidence indicates that pulmonary exposure to CNTs induces lung fibrosis, a fetal and incurable lung disease with no known effective treatments. The long-term objective of this project is to enable safe nanotechnology through the understanding of underlying mechanisms of fibrogenesis and determining key physicochemical factors controlling the fibrogenic effect of nanomaterials. Evolving research indicates that fibroblast stem cells (FSCs) with unlimited proliferative potential are likely a driing force of fibrosis, but the underlying mechanisms and their role in CNT fibrogenesis are not known. We have obtained breakthrough evidence showing the ability of CNTs to induce FSCs with a functional phenotype of activated fibroblasts that are responsible for extracellular matrix accumulation, a hallmark of lung fibrosis. We hypothesize that CNTs induce lung fibrosis through a process that involves FSC induction and that such induction is dependent on specific physicochemical properties of CNT. Three specific aims are proposed to test the hypotheses. In Aim 1, we will characterize FSC acquisition in CNT-treated fibroblasts and animals and determine their role in fibrogenesis. Aim 2 will develop 3D high-throughput fibroblastic nodule models for quantitative assessment of CNT fibrogenicity and document the impact of CNT characteristics on FSC acquisition and fibrogenesis. This Aim will also identify specific FSC biomarkers and validate the in vitro models in animals. Aim 3 will examine redox regulation of FSC development and fibrogenesis, and determine specific oxidative species and key regulatory enzymes involved in the process. Our expectations are that at the conclusion of this project, we will have determined the role of FSCs in CNT fibrogenesis and identified specific biomarkers and key physicochemical properties of CNTs that influence their fibroticity. This work is important because of the overall impact it will have on the development of safe nanomaterials as well as on risk assessment, early detection and prevention of nanomaterial-induced fibrosis. We expect this impact to be broad since the findings from this project are highly applicable to other fibrogenic agents, nanomaterials and xenobiotics. The proposed work is innovative because it is the first to study FSCs and their role in fibrosis. It will also develop novel experimental models and assay methods for rapid assessment of nanomaterial fibrogenicity and for their potential utility in various stem cell applications.